Mass spectrometry (MS) is often utilized to characterize large (high molecular-weight) molecules including long-chain biopolymers (e.g., peptides, proteins, etc.). In the simplest typical work flow, intact large molecules are separated, ionized, and introduced to a mass spectrometer where the ion mass-to-charge (m/z) ratio is measured and utilized to deduce molecular formulae. In tandem mass spectrometry (MS/MS), additional information is gained by expanding the workflow to include a fragmentation step in which an ion or ions of interest (“precursor” or “parent” ions) are isolated by m/z ratio and then dissociated (fragmented) into smaller “product” or “fragment” ions. The fragment masses offer complementary molecular information and consequently play an important role in characterizing large molecules in situations where the mass measurement alone is inadequate.
Numerous fragmentation methods exist, each with its own merits and disadvantages. The mechanism for dissociation usually performed in a Paul trap or other type of radio frequency (RF) based ion processing device is collision-induced dissociation (CID), also referred to as collision-activated dissociation (CAD). CID entails accelerating a parent ion to a high kinetic energy in the presence of a background neutral gas (or collision gas) such as helium, nitrogen or argon. When the excited parent ion collides with the gas molecule, some of the parent ion's kinetic energy is converted into internal (vibrational) energy. If the internal energy is increased high enough, the parent ion will break into one or more fragment ions, which may then be mass-analyzed. A similar mechanism is employed in Penning traps, known as sustained off-resonance irradiation (SORI) CID, which entails accelerating the ions so as to increase their radius of cyclotron motion in the presence of a collision gas. An alternative to CID and SORI-CID is infrared multiphoton dissociation (IRMPD), which entails using an IR laser to irradiate the parent ions whereby they absorb IR photons until they dissociate into fragment ions. IRMPD is also based on vibrational excitation (VE).
CID and IRMPD are not considered to be optimal techniques for dissociating ions of large molecules such as peptides and proteins. For many types of large molecules these VE-based techniques are not able to cause the types of bond cleavages, or a sufficient number of these cleavages, required to yield a complete structural analysis. Currently, electron capture dissociation (ECD) is being investigated as a promising new method for dissociating large molecular ions. In ECD, the well-known technique of electrospray ionization (ESI) is usually selected to produce positive, multiply-charged ions of large molecules by proton attachment. The “soft” or “gentle” technique of ESI leaves the multiply-charged ions intact, i.e., not fragmented. The ions are then irradiated by a stream of low-energy free electrons. If their energy is low enough (typically less than 3 eV), the electrons can be captured by the positively charged sites on the ions. The energy released in the exothermic capture process is released as internal energy in the ion, which can then very quickly cause bond cleavage (at a peptide backbone, for example) and dissociation. ECD is considered to be a particularly powerful method for fragmenting intact proteins and large peptides. The advantages of ECD are that the fragmentation pattern is simple and predictable, which aids in protein identification, and post-translation modifications of the amino acid residues are kept intact throughout the fragmentation process.
State of the art ECD systems use heated cathode filaments as the source of electrons, which are liberated from the filament surfaces by thermionic emission. This type of device is commonly used in conjunction with “hard” electron impact (EI) ionization and other processes requiring the production of an intense electron beam. To reach high electron thermionic emission currents, the filaments are heated to at least several hundred degrees Kelvin, which heats the wires delivering the filament current as well as the surrounding system. The magnetic fields generated by the filament current as well as electric field from the voltage drop across the filament must also be considered in the design. Additionally, the high extraction voltage required to form an electron beam from a heated filament surface produces high energy electrons, which are not suitable for ECD as noted above. Moreover, when the filament is operating at the space-charge limit for the low electron energies (less than 2 eV), the electron density is low, resulting in either low efficiency or requiring very long interaction distances and times. In the state of the art ECD mass spectrometers based on magnetic trapping (i.e., Fourier transform ion-cyclotron resonance MS), the low electron density is offset by long interaction distances and times. The resulting system does not have high throughput and does not operate on a time-scale compatible with modern chromatographic separations.
As an alternative to an electron beam produced by thermionic emission, plasma can serve as an excellent source of a high-density population of electrons. However, there are a number of other species of particles present in plasma. In a plasma for which the gas employed is a noble gas, the most important of these species are: (1) plasma electrons—free electrons created by ionizing collisions, which exhibit a range of energies; (2) plasma ions—positively charged ions created in the same ionizing collisions; (3) metastable atoms—neutral atoms that have stored energy in a long-lived metastable state as a result of non-ionizing collisions; (4) ultraviolet (UV) photons—UV light generated by the collisional excitation and decay of atoms; and (5) neutral atoms—unexcited neutral atoms, typically at a density much higher than all other species. Of all of these species, only low-energy (less than 3 eV) plasma electrons meet the requirements for successful fragmentation of analyte parent ions through the mechanism of ECD. High-energy plasma electrons and all other species are undesirable as they may cause unwanted ionization or dissociation events that serve only as background noise in the resulting mass spectrum.
Therefore, there is a need for plasma-based ECD apparatuses and methods. There is also a need for plasma-based ECD apparatuses and methods capable of removing unwanted plasma species from the plasma. There is also a need for plasma-based ECD apparatuses and methods capable of producing optimal densities of low-energy plasma electrons.